Runsheng Li

Microvesicle-Shuttled miR-130b Reduces Fat Deposition in Recipient Primary Cultured Porcine Adipocytes by Inhibiting PPAR-γ Expression

Obesity is a worldwide epidemic, and a risk factor for cardiovascular disease and type 2 diabetes. Consequently, the development of safe and effective anti‐obesity drugs is an area of ongoing clinical interest. MicroRNAs play a vital role in anti‐obesity by inhibiting the expression of genes involved in adipogenesis and lipogenesis. However, the clinical application of miRNAs has been limited by a lack of appropriate delivery systems. The discovery of microvesicles (MVs) has shed new light on the search for more efficient drug transport tools. In a previous study, we demonstrated that miRNA‐130b suppressed fat deposition by inhibiting PPAR‐γ expression. In order to demonstrate whether miRNA‐130b can be packaged into MVs and function as an endogenous form of miRNA‐130b in recipient cells, we transfected HeLa‐229 cells with plasmid to overexpress miRNA‐130b. This enabled HeLa‐229 cells to selectively package miRNA‐130b into MVs and actively secrete the miRNA‐130b enriched MVs into the culture media. We further verified that MVs enriched with miRNA‐130b contain elevated concentrations of Argonaute 2 and heat shock protein 90α which are known to protect the circulating miRNAs from degradation. Exposure of primary cultured porcine adipocytes to purified, miRNA‐130b‐enriched MVs resulted in a significant down‐regulation of PPAR‐γ expression which was associated with reduced adipogenesis and lipogenesis. Taken together, our results suggest that MVs may provide an effective transport systems for the deliver of miRNAs for therapeutic use. We also showed that MV‐shuttled miRNA‐130b inhibited adipogenesis and lipogenesis, and reduced fat deposition in recipient adipocytes by targeting PPAR‐γ. J. Cell. Physiol. 229: 631–639, 2014.

Maternal dietary protein affects transcriptional regulation of myostatin gene distinctively at weaning and finishing stages in skeletal muscle of Meishan pigs

Myostatin (MSTN) is suggested to mediate the effect of maternal nutrition on offspring phenotype, yet the mechanisms underlying such adaptive gene regulation is elusive. In this study, we determined the effects of maternal dietary protein on transcriptional regulation of MSTN in skeletal muscle of pig offspring. Fourteen Meishan sows were fed either low-protein (LP) or standard-protein (SP) diets throughout gestation and lactation. MSTN expression in the longissimus dorsi muscle was determined both at weaning and finishing stages. Myostatin mRNA abundance was downregulated at weaning, but upregulated at finishing in LP pigs, indicating stage-specific transcriptional regulation. At weaning, CCAAT/enhancer-binding protein beta (C/EBPβ) in muscle nuclear lysate was decreased in LP piglets, associated with diminished binding of C/EBPβ to all the 3 putative binding sites in MSTN promoter. None of the four histone modification marks investigated showed differences between SP and LP piglets. Among 12 microRNAs predicted to target MSTN, none was differently expressed. At finishing stage, C/EBPβ content remained unchanged, but the binding of C/EBPβ to two of the 3 putative binding sites increased in LP pigs. Histone H3 acetylation and histone H3 lysine 27 trimethylation on MSTN promoter were increased, while histone H3 lysine 9 monomethylation was decreased in LP pigs. Moreover, expression of ssc-miR-136 and ssc-miR-500 was significantly reduced. These results indicate that maternal dietary protein affects MSTN expression through distinct regulatory mechanisms at different stages. The immediate effect at weaning is mediated by C/EBPβ binding without epigenetic modifications, whereas the long-term effect at finishing stage involves both C/EBPβ binding and epigenetic regulations, including histone modification and microRNA expression.

Maternal Low-Protein Diet Affects Epigenetic Regulation of Hepatic Mitochondrial DNA Transcription in a Sex-Specific Manner in Newborn Piglets Associated with GR Binding to Its Promoter

Mitochondrial oxidative phosphorylation (OXPHOS) plays an important role in energy homeostasis by controlling electron transfer and ATP generation. Maternal malnutrition during pregnancy affects mitochondrial (mt) DNA-encoded OXPHOS activity in offspring, yet it is unknown whether epigenetic mechanism is involved in the transcriptional regulation of mtDNA-encoded OXPHOS genes. In this study, 14 primiparous purebred Meishan sows were fed either standard- (SP, 12% crude protein) or low-protein (LP; 6% crude protein) diets throughout gestation, and the hepatic expression and transcriptional regulation of mtDNA-encoded OXPHOS genes were analyzed in newborn piglets. Maternal low protein diet decreased hepatic mtDNA copy number in males, but not in females. LP male piglets had significantly higher hepatic AMP concentration and low energy charge, which was accompanied by enhanced mRNA expression of NADH dehydrogenase subunits 6, cytochrome c oxidase subunit 1, 2, 3 and cytochrome b, as well as increased cytochrome c oxidase enzyme activity. In contrast, LP female piglets showed significantly lower hepatic AMP concentrations and higher energy charge with no alterations in OXPHOS gene expression. Moreover, LP males demonstrated higher glucocorticoid receptor (GR) binding to the mtDNA promoter compared with SP males, which was accompanied by lower cytosine methylation and hydroxymethylation on mtDNA promoter. Interestingly, opposite changes were seen in females, which showed diminished GR binding and enriched cytosine methylation and hydroxymethylation on mtDNA promoter. These results suggest that maternal low protein diet during pregnancy causes sex-dependent epigenetic alterations in mtDNA-encoded OXPHOS gene expression, possibly GR is involved in mtDNA transcription regulation.

Coordinated miRNA/mRNA Expression Profiles for Understanding Breed-Specific Metabolic Characters of Liver between Erhualian and Large White Pigs

MicroRNAs (miRNAs) are involved in the regulation of various metabolic processes in the liver, yet little is known on the breed-specific expression profiles of miRNAs in coordination with those of mRNAs. Here we used two breeds of male newborn piglets with distinct metabolic characteristics, Large White (LW) and Erhualian (EHL), to delineate the hepatic expression profiles of mRNA with microarray and miRNAs with both deep sequencing and microarray, and to analyze the functional relevance of integrated miRNA and mRNA expression in relation to the physiological and biochemical parameters. EHL had significantly lower body weight and liver weight at birth, but showed elevated serum levels of total cholesterol (TCH), high-density lipoprotein cholesterol (HDLC) and low-density lipoprotein cholesterol (LDLC), as well as higher liver content of cholesterol. Higher serum cortisol and lower serum insulin and leptin were also observed in EHL piglets. Compared to LW, 30 up-regulated and 18 down-regulated miRNAs were identified in the liver of EHL, together with 298 up-regulated and 510 down-regulated mRNAs (FDR<10%). RT-PCR validation of some differentially expressed miRNAs (DEMs) further confirmed the high-throughput data analysis. Using a target prediction algorithm, we found significant correlation between the up-regulated miRNAs and down-regulated mRNAs. Moreover, differentially expressed genes (DEGs), which are involved in proteolysis, were predicted to be mediated by DEMs. These findings provide new information on the miRNA and mRNA profiles in porcine liver, which would shed light on the molecular mechanisms underlying the breed-specific traits in the pig, and may serve as a basis for further investigation into the biological functions of miRNAs in porcine liver.

Breed-Dependent Transcriptional Regulation of 5′-Untranslated GR (NR3C1) Exon 1 mRNA Variants in the Liver of Newborn Piglets

Glucocorticoids are vital for life and regulate an array of physiological functions by binding to the ubiquitously expressed glucocorticoid receptor (GR, also known as NR3C1). Previous studies demonstrate striking breed differences in plasma cortisol levels in pigs. However, investigation into the breed-dependent GR transcriptional regulation is hampered by lacking porcine GR promoter information. In this study, we sequenced 5.3 kb upstream of the translation start codon of the porcine GR gene, and identified seven alternative 5′-untranslated exons 1–4, 1–5, 1–6, 1–7, 1–8, 1–9,10 and 1–11. Among all these mRNA variants, exons 1–4 and 1–5, as well as the total GR were expressed significantly (P<0.05) higher in the liver of newborn piglets of Large White (LW) compared with Erhualian, a Chinese indigenous breed. Overall level of CpG methylation in the region flanking exons 1–4 and 1–5 did not show breed difference. However, nuclear content of Sp1, p-CREB and GR in the liver was significantly (P<0.05) higher in LW piglets, associated with enhanced binding of p-CREB, and higher level of histone H3 acetylation in 1–4 and 1–5 promoters. In contrast, GR binding to promoters of exons 1–4 and 1–5 was significantly diminished in LW piglets, implicating the presence of negative GREs. These results indicate that the difference in the hepatic expression of GR transcript variants between two breeds of pigs is determined, at least partly, by the disparity in the binding of transcription factors and the enrichment of histone H3 acetylation to the promoters.

Maternal protein restriction during gestation and lactation programs offspring ovarian steroidogenesis and folliculogenesis in the prepubertal gilts

Maternal malnutrition may disrupt ovarian functions in adult offspring. Steroidogenesis and folliculogenesis in the offspring ovary appear to be the major targets of nutritional programming. Nevertheless, the mechanism by which maternal low-protein diet affects the offspring steroidogenesis and folliculogenesis, and the possible pathway linking these two processes remain unclear. In this study, Landrace×Yorkshire crossbred sows were fed either standard (SP) or low-protein (LP, 50% of the SP) diets throughout gestation and lactation. Female offspring were fed the same diet after weaning until 6 months of age. LP offspring had higher serum 17β-estradiol level (P<0.01), which was accompanied by lower mRNA (P<0.05) but higher protein (P<0.05) expression of cytochrome P450 aromatase (CYP19A1) in the ovary. CYP19A1 protein up-regulation was associated with lower ovarian expression of drosha (P<0.05) and miRNAs targeting CYP19A1 (P<0.05). LP offspring had less graafian follicles with more apoptotic granulosa cells (P<0.05), as well as higher caspase 3 activity (P<0.05) and FasL expression (P<0.05) in the ovary. FasL gene up-regulation was associated with higher ERα protein expression (P<0.05) and binding to FasL gene promoter. These results suggest that a maternal LP diet in pregnancy and lactation elevated serum 17β-estradiol level by activating CYP19A1 through miRNA-mediated mechanism, and induced granulosa apoptosis in graafian follicles through ER-activated Fas/FasL-caspase 3 pathway.

Maternal Low-protein Diet Alters Ovarian Expression of Folliculogenic and Steroidogenic Genes and Their Regulatory MicroRNAs in Neonatal Piglets

Maternal malnutrition during pregnancy may give rise to female offspring with disrupted ovary functions in adult age. Neonatal ovary development predisposes adult ovary function, yet the effect of maternal nutrition on the neonatal ovary has not been described. Therefore, here we show the impact of maternal protein restriction on the expression of folliculogenic and steroidogenic genes, their regulatory microRNAs and promoter DNA methylation in the ovary of neonatal piglets. Sows were fed either standard-protein (SP, 15% crude protein) or low-protein (LP, 7.5% crude protein) diets throughout gestation. Female piglets born to LP sows showed significantly decreased ovary weight relative to body weight (p<0.05) at birth, which was accompanied with an increased serum estradiol level (p<0.05). The LP piglets demonstrated higher ratio of bcl-2 associated X protein/B cell lymphoma/leukemia-2 mRNA (p<0.01), which was associated with up-regulated mRNA expression of bone morphogenic protein 4 (BMP4) (p<0.05) and proliferating cell nuclear antigen (PCNA) (p<0.05). The steroidogenic gene, cytochrome P450 aromatase (CYP19A1) was significantly down-regulated (p<0.05) in LP piglets. The alterations in ovarian gene expression were associated with a significant down-regulation of follicle-stimulating hormone receptor mRNA expression (p<0.05) in LP piglets. Moreover, three microRNAs, including miR-423-5p targeting both CYP19A1 and PCNA, miR-378 targeting CYP19A1 and miR-210 targeting BMP4, were significantly down-regulated (p<0.05) in the ovary of LP piglets. These results suggest that microRNAs are involved in mediating the effect of maternal protein restriction on ovarian function through regulating the expression of folliculogenic and steroidogenic genes in newborn piglets.

Sexually dimorphic effects of maternal dietary protein restriction on fetal growth and placental expression of 11β-HSD2 in the pig

Placental 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2) inactivates glucocorticoids (GCs) to protect fetuses from over-exposure to maternal GCs, yet how maternal malnutrition affects placental 11β-HSD2 expression is unknown. In this study, Meishan sows were fed standard-protein (SP) or low-protein (LP, 50% of SP) diets and fetuses/newborn piglets were weighed and the corresponding placenta and umbilical cord blood were collected on gestational day 70 and the day of parturition. Significant growth retardation was observed in female, but not male, fetuses (P < 0.05) and the newborns (P < 0.01) of the LP group, which was accompanied by sexually dimorphic expression of 11β-HSD2 in placentas. Female fetuses in LP group showed significant decrease in placental 11β-HSD2 protein content (P < 0.05) and enzyme activity (P < 0.05), whereas male fetuses demonstrated significantly enhanced placental 11β-HSD2 activity (P < 0.05). Serum cortisol levels were significantly higher (P < 0.05) in male piglets compared to females, and the effects of maternal protein restriction on thyroid hormones (T3 and T4) in the umbilical cord blood were also sex dimorphic. Male piglets in LP group had significantly higher T3 (P < 0.01) and lower T4 (P < 0.01), whereas female piglets showed significantly lower T4 (P < 0.01) with no change in T3. As a result, male piglets in LP group exhibited significantly higher T3/T4 ratio compared to female counterparts. These results indicate that the effects of maternal protein restriction on placental 11β-HSD2 expression are gender-dependent in the pig, and thyroid hormones may be involved in such effects.

Breed-specific expression of GR exon 1 mRNA variants and profile of GR promoter CpG methylation in the hippocampus of newborn piglets.

Glucocorticoid receptor (GR) transcription is driven by alternative promoters to produce different exon 1 mRNA variants. CpG methylation on GR promoters profoundly affects GR transcription. GR in hippocampus is critical for energy homeostasis and stress responses, yet it remains unclear whether hippocampal expression of GR exon 1 mRNA variants and the methylation status of GR promoters differ between Large White (LW) and Erhualian (EHL) pigs showing distinct metabolic and stress-coping characteristics. EHL pigs had higher hippocampus weight relative to BW (P<0.01), which was associated with higher serum cortisol level compared with LW pigs. Hippocampal expression of brain-derived neurotrophic factor (P<0.05) was significantly higher, while Bax, a pro-apoptotic gene, was significantly lower in EHL pigs (P<0.05). Hippocampal expression of total GR did not differ between breeds, yet GR exon 1 to 11 mRNA was significantly higher (P<0.01) in EHL pigs, which was associated with a trend of increase (P=0.057) in GR protein content. No significant breed difference was detected for the methylation status across the whole region of the proximal GR promoter, while CpG334 and CpG266.267 were differentially methylated, in a reversed manner, between breeds. The methylation status of CpGs 248, 259, 260, 268 and 271 was negatively correlated (P<0.05) with GR exon 1 to 11 mRNA abundance. Our results provide fundamental information on the breed-specific characteristics of GR and its mRNA variants expression and the status of DNA methylation on the proximal GR promoter in the pig hippocampus.

p53 cooperates with Sp1 to regulate breed-dependent expression of glucocorticoid receptor in the liver of preweaning piglets.

Previous studies indicate that Chinese indigenous pig breeds demonstrate distinct pattern of glucocorticoid receptor (GR) expression, which is associated with their unique growth and metabolic phenotypes. Here we sought to unravel the transcriptional mechanisms underlying the breed-specific hepatic GR expression in preweaning Chinese Erhualian (EHL) and Western Large White (LW) piglets. Total GR mRNA and the predominant GR mRNA variant 1–9/10 were expressed significantly higher in EHL compared with LW piglets (P<0.01), which was associated with more enriched histone H3 acetylation on 1–9/10 promoter (P<0.05). Nuclear content of transcription factor specificity protein 1 (Sp1) was significantly lower in EHL piglets, yet its binding to GR 1–9/10 promoter was significantly higher in EHL piglets, as revealed by chromatin immunoprecipitation assays. Although p53 binding to GR promoter 1–9/10 did not differ between breeds, expression of p53 mRNA and protein, as well as its binding to Sp1, were significantly higher in EHL piglets. Moreover, p53 activator doxorubicin significantly enhanced GR 1–9/10 promoter activity in HepG2 cells at 100 nM, which was associated with significantly higher protein content of p53 and GR. Sp1 inhibitor, mithramycin A, significantly inhibited (P<0.05) the basal activity of GR promoter 1–9/10 and completely blocked doxorubicin -induced activation of GR promoter 1–9/10. These data indicate that higher hepatic GR expression in EHL piglets attributes mainly to the enhanced transcription of GR promoter 1–9/10, which is achieved from breed-specific interaction of p53 and Sp1 on porcine GR 1–9/10 promoter.